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FP7

HighGlycan Report Summary

Project ID: 278535
Funded under: FP7-HEALTH
Country: Netherlands

Periodic Report Summary 3 - HIGHGLYCAN (Methods for high-throughput (HTP) analysis of protein glycosylation)

Project Context and Objectives:
The HighGlycan project (http://www.highglycan.eu/) develops technology for the analysis of protein glycosylation in a high-throughput (HTP) manner. Protein glycosylation plays an important role in determining the stability of proteins. Glycosylation targets proteins to specific subcellular compartments or – in the case of secreted proteins – to specific sites in the organism. Changes in glycan structure modulate the activity of proteins, by influencing for example receptor-ligand interactions. Protein glycosylation is highly regulated, and cellular development steps consistently coincide with glycosylation changes. Glycosylation patterns may mark various pathological and physiological situations. Diseases associated with glycosylation changes include cancer and diabetes, as well as congenital, cardiovascular, immunological and infectious disorders. Various glycoproteins have been found to be useful clinical biomarkers in diagnostics as well as targets for disease therapy.

There is an increasing need in both biomedical research and biotechnology for robust high-throughput methods for the characterization of protein glycosylation. The HighGlycan consortium addresses this need by developing analytical methods which can reliably quantify a multitude of glycan structures in complex biological samples. For this purpose, it brings together leading European companies as well as innovative academic groups in the field of high-throughput glycosylation analysis.
On the basis of analytical potential as well as acceptance in the field, the HighGlycan participants have chosen the following three technologies for further development of advanced, mature glycosylation profiling methods:
1. Ultrahigh-performance liquid chromatography (UPLC)
2. Capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF)
3. Mass spectrometry (MS)
The HighGlycan consortium works on method development including robotisation of sample preparation methods. Another focus is on data processing and analysis methods. In addition, method comparison is an important aspect of the activities, in order to evaluate the particular strengths and limitations of the different analytical approaches. The developed methods show a considerable overlap in terms of the analytical information they provide, but also exhibit divergent properties with regard to throughput as well as required expertise and investments.

Strong participation of SMEs in the project and close contacts with large industrial partners will ensure that research accomplishments achieved by collaboration between academic and industrial scientists are swiftly transformed into innovative products and services for the benefit of European industry. Moreover, close association with clinical research initiatives ensures the efficient transfer of the developed technology into biomarker discovery programs. Due to the high-throughput of the developed glycoanalytical techniques, novel approaches such as genome-wide association studies of glycomic profiles can be performed, providing new insights into the (dys)regulation of glycosylation in human development and disease settings. Additionally, by combining glycomics information with other ‘omics’ and phenotypic data, glycomics will be integrated into systems biology type studies.

Project Results:
Sample preparation for high-throughput glycomics.
A vast part of the work within HighGlycan was hitherto focused on the development of suitable sample preparation methods and derivatisation techniques for the analysis of protein N-glycosylation (see mainly WP2 and WP3, but also WP4 to 6). An important aspect was the search for sample preparation techniques which excel with regard to sensitivity, resolution, simplicity and robustness. In addition, robotisation of protocols got considerable attention.
The consortium has achieved very good progress with regard to robotisation and automation of glycomic sample preparation methods. This resulted in several largely automated glycomic workflows for UPLC profiling of protein glycosylation, but also for mass spectrometry. All the workflows showed a remarkable robustness. Notably, next to the increase in throughput, the increase in robustness when moving from (different) persons doing manual sample preparation to a robotic platform proved to be remarkable. Very recently, also several workflows for the mass spectrometric analysis of protein glycosylation were transferred to robotic platforms.
The established workflows have been transferred between laboratories within the HighGlycan consortium for further evaluation and comparison of the workflows. Currently, successful HighGlycan technology is being disseminated to academic research laboratories as well as the biopharmaceutical industry.

Improvement of glycomics measurement techniques.
Several improvements have been achieved with regard to the high-throughput measurement techniques: for the UPLC analysis of glycans, a multiplexed method was established which gives the potential of directly comparing two samples in a single run, as well as increasing sample throughput (WP4). Importantly, new labels were implemented that excel by higher sensitivity in both mass spectrometric and fluorescence detection. Some of the new labels rely on different coupling chemistry, thereby allowing faster and more facile labelling of glycans. For the CGE-LIF measurement method, major progress was achieved by increasing the set of available standards and improving the data analysis tools, both supporting glycan structural assignment (WP5). For the mass spectrometric analysis, robotization was achieved, as well as standardization and automation of data processing with integrated workflows (WP6). Importantly, we performed an initial comparison of the current-stage high-throughput glycoanalytical techniques (WP7). These analyses were performed on approximately 1800 samples that were analysed with four different analytical approaches (UPLC, CGE-LIF, as well as two mass spectrometric methods). The major evaluation criterion was whether the methods revealed genome-wide associations (GWA) of single nucleotide polymorphisms with glycomic profiles and features. Importantly, all four methods revealed largely consistent sets of genetic associations, thereby unambiguously establishing the potential of all these methods in high-throughput biomedical research.
Additional method comparison studies are currently going on within HighGlycan covering newly developed robotised workflows. In addition, more complex and demanding challenges were chosen for method comparison, in order to evaluate method performance for adanced applications. These applications cover both biotechnology and clinical areas.

Valorisation of technology by SMEs and industry.
It is an important aim of the HighGlycan project to ensure that the developed technology finds a place in biotechnological applications (WPs 9 and 10). Some of the HighGlycan technique has already found its way into industrial glycoanalytical services via SMEs participating in the project. Likewise, Ludger has already launched glycoanalytical kits based on technology developed within the HighGlycan project, and more kits are to follow. In the same line, NIBRT is successfully providing UPLC technology to biotech and pharma companies. Part of the technology developed within HighGlycan is comprised by a patent application which will be exploited by the HighGlycan beneficiary GlyXera.

Dissemination of technology into biomedical research.
Application of the developed high-throughput technology is part of the project (WPs7 and 8), and some important results have already been achieved by glycomics GWAS analyses that revealed important aspects of the regulation of the glycosylation of the proteins in the human circulation. WP8 will apply the technique to study protein glycosylation changes in diabetes. Application of the technology beyond the HighGlycan project is ensured by various research projects that are using the developed high-throughput glycomics methods, one prominent example being the IBD-BIOM project (www.ibdbiom.eu) dealing with the search for diagnostic biomarkers and therapeutic targets of inflammatory bowel disease. Importantly, efficient data analysis of glycomics data in a systems biology approach as pursued in WP7 is strongly supported by close interaction with the MIMOmics consortium (http://www.mimomics.eu/) that develops methods for ‘omics’ metadata analysis.
The use of HighGlycan technology is further boosted in various new biomedical and clinical research projects such as the GlyCoCan ITN (https://glycocan.eu/) which started in 2015, and the GlySign European Industrial Doctorate programme (EID) project which has been selected for funding and is scheduled to start in October 2016.

Potential Impact:
Protein glycosylation is critically important for proper functioning of natural biological systems as well as biopharmaceuticals and vaccines. However, current glycoanalysis tools fail to deliver the analytical power and high sample throughput required for studying the human glycome or efficiently dealing with drug glycosylation. This failure results both from the immense structural complexity of glycosylation and the lack of development resources for high-throughput (HTP) glycoprofiling tools.
We expect the HighGlycan program to fill that technology gap through development of relevant, reliable, and affordable commercial HTP glycoprofiling systems for two application areas:
a. Glycomics studies for systems biology and biomedical research
b. Glycan characterisation for improvement of glycoprotein therapeutics.
The program will provide means for reliable identification and quantitation of glycans and achieve high-throughput analysis by multiplexing and automation. A flexible, modular glycoprofiling system will be developed based on three orthogonal analytical platforms (UPLC, MS and CGE-LIF) with proven utility for detailed, quantitative glycoprofiling. Commercial-grade technology will be developed that will be both used and sold by the participants, over half of which are SMEs specialising in products and services for glycoanalysis.
We expect the HighGlycan program to have a significant, positive impact on the project's participants, the clients of the HTP glycoprofiling technology, and ultimately patients benefiting from the diagnostics and disease treatments that are enabled by that technology.

List of Websites:
http://www.highglycan.eu/

Contact

Manfred Wuhrer, (Associate Professor)
Tel.: +31715265081
Fax: +31715266907
E-mail

Subjects

Life Sciences
Record Number: 189392 / Last updated on: 2016-09-20
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